A litchi downy mildew resistance gene LcCLP1 and its encoding protein and application

Abstract

The invention discloses a litchi downy mildew disease resistance gene LcCLP1 and a coding protein and application, and relates to the field of bioengineering technology. The technical scheme thereof is as follows: selecting fruits of litchi varieties that are stable susceptible to downy mildew and litchi varieties that are resistant to disease as samples; using transcriptome sequencing analysis to obtain a batch of differentially expressed genes from samples at different time points after downy mildew infection; obtaining a disease resistance-related gene CLP from the differentially expressed genes obtained in step S2; cloning the full length of the gene sequence based on transcriptome data; and verifying the relative expression level of the LcCLP1 gene by qRT-PCR. The gene has broad-spectrum resistance. If the creation of disease-resistant resources is used, the overexpression of a gene can achieve the effect of defending against two pathogens (litchi anthracnose and downy mildew); the gene has broad-spectrum resistance, and in molecular marker-assisted breeding, the workload can be reduced by 50% when disease-resistant resources are selected in the early stage.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and more specifically to a litchi downy mildew disease-resistant gene LcCLP1, a coded protein and an application thereof. Background Art

[0002] Litchi is an important fruit tree in southern my country and occupies an important position in the agricultural industry economy. However, during production, litchi is easily attacked by various diseases and pests, such as litchi anthracnose and litchi downy mildew. Litchi anthracnose can occur throughout the tree throughout the year, causing leaf death and affecting photosynthesis, flower spikes and young fruits to fall off, affecting flowering and fruiting, and it is easy to break out on a large scale during the storage and transportation of fruits after harvest, seriously affecting the storage period and shelf life of the fruits. Litchi downy mildew generally occurs in fruit spikes and mature fruits, affecting flowering and fruiting, and occurs on post-harvest fruits, causing browning and rotting of the fruits. The damage caused by litchi anthracnose and downy mildew is the main cause of post-harvest litchi fruit rot.

[0003] At present, fungicides are mostly used to prevent and control diseases during growth, mainly chemical fungicides. Long-term use can easily lead to increased drug resistance of diseases, environmental pollution, pesticide residues in fruits and other problems. Using resistance genes to carry out biotechnology breeding is the main method to solve diseases, and it is also a long-term and effective method. The screening of key disease-resistant genes is one of the keys.

[0004] Through years of research, we have discovered a stable litchi resource 'Guiwei' that is highly susceptible to downy mildew and a resource 'Heiye' that is highly resistant (Sun et al., 2019). Using this as a control, we conducted transcriptome sequencing analysis of the pericarp after downy mildew infection (Sun et al., 2019), and obtained a number of differentially expressed genes (Sun et al., 2019). Among them, a Curculin-like mannose-binding lectin family protein (CLP) gene was upregulated in the pericarp of the resistant resource in response to the invasion of pathogens. Studies in Arabidopsis have found that this gene plays a role in signal transduction. Summary of the invention

[0005] The purpose of the present invention is to provide a litchi downy mildew resistance gene LcCLP1 and its encoding protein and application, and to analyze the function of the gene in litchi disease resistance, so as to use it for subsequent molecular assisted breeding and improve breeding efficiency.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A litchi downy mildew resistance gene LcCLP1 and its encoding protein and application, comprising the following steps: S1: selecting fruits of a susceptible litchi variety that is stably susceptible to downy mildew and a resistant litchi variety that is resistant to downy mildew as samples;

[0007] S2: Using transcriptome sequencing to analyze samples at different time points after infection with Downy Phytophthora, a group of differentially expressed genes were obtained;

[0008] S3: obtaining a disease resistance-related gene CLP from the differentially expressed genes obtained in step S2; performing transcription, and based on the transcriptome data, cloning the full-length gene sequence and naming it LcCLP1;

[0009] S4: The relative expression level of the LcCLP1 gene was verified by qRT-PCR using the peels of disease-resistant and susceptible litchi varieties after inoculation.

[0010] The present invention is further configured as follows: the differentially expressed gene described in step S3 has a higher expression level in the pericarp of varieties resistant to downy mildew, and a lower expression level in the pericarp of varieties susceptible to the disease.

[0011] The present invention is further configured as follows: in the step S1, the disease-susceptible litchi variety is the 'Guiwei' litchi variety, and the disease-resistant litchi variety is the 'Heiye' litchi variety.

[0012] In summary, the present invention has the following beneficial effects: 1. The gene has broad-spectrum resistance. If the creation of disease-resistant resources is utilized, the overexpression of one gene can achieve the effect of defending against two pathogens (litchi anthracnose and downy mildew); 2. The gene has broad-spectrum resistance. In molecular marker-assisted breeding, the workload can be reduced by 50% when disease-resistant resources are selected in the early stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a graph of transcriptome analysis results in an embodiment of the present invention;

[0014] Figure 2 is the relative expression level of the LcCLP1 gene in the litchi leaves inoculated with anthrax fungi in the embodiment of the present invention;

[0015] Figure 3 is the result of nucleic acid sequence comparison of CLP genes of four litchi resources in the embodiment of the present invention;

[0016] Figure 4 It is the result of amino acid sequence comparison of CLP genes of 4 litchi resources in the example of the present invention. DETAILED DESCRIPTION

[0017] The following is combined with Figure 1-4 The present invention is described in further detail.

[0018] Embodiment: A litchi downy mildew resistance gene LcCLP1 and its encoding protein and application, such as Figure 1 , Figure 2As shown, the fruits of the varieties 'Guiwei', which are stably susceptible to downy mildew, and 'Heiye', which is resistant, were used as materials. Samples at different time points after pathogen infection were analyzed by transcriptome sequencing to obtain a batch of differentially expressed genes (Sun et al., 2019).

[0019] Among the differentially expressed genes, a disease resistance-related gene CLP (Curculin-like mannose-binding lectin family protein, CLM) was found, and its expression level was higher in the peel of varieties resistant to downy mildew, and lower in the peel of varieties susceptible to the disease.

[0020] Among the differentially expressed genes, the expression level of the CLP gene (Litc.03B008940) in the disease-resistant variety 'Black Leaf' increased rapidly in the early stage (6 h after inoculation), then decreased, and continued to increase from 48 to 72 h after inoculation ( Figure 1 ). The expression level in the susceptible variety 'Guiwei' remained low, and it was initially predicted to be a disease resistance-related gene.

[0021] Based on the transcriptome data, the full-length gene sequence was cloned and named LcCLP1. The full-length gene sequence is 1356 bp and encodes 451 amino acids. The gene sequence of gene LcCLP1 is:

[0022] atggctgctt ttcatgtaat tttagtccat agcaccctcc tcagcctagc catcaccgga

[0023] actctatccc aagtcccaac taaccaaacc ttcaaattca taaaccaagg tgaattcgga

[0024] gacagaataa tcgaatatga cgccagttac cgtgtgatcc gaaacaatgt ttacacgttc

[0025] tacacattcc catttcgcct gtgtttctac aacaccaccc ctaatgccta catttttgct

[0026] attagggcag gcatccctaa tgatgaaagc ctcatgcgct gggtttggga cgccaaccgc

[0027] aacaacccgg tacgtgaga tgccaccctc acctttggcc cttggtcctg

[0028] gcggccgccg atggtcgcgt ggcctggcaa acaaacacgg ccaacaaggg ggtcactggc

[0029] atcaagttgt taccaatgg taacctagtg ttgcatgaca aaaatgggaa atttatatgg

[0030] gaaagttttg attaccctag tgatacttta ttggtgggcc agtcagtcaa aatcaacggt

[0031] cgtaacaaac ttgttggtcg tacgtctgac atgattggct ctgatggacg gtacagtatt

[0032] gtgctggatc ataaagggtt caccatgtat ttaaatatt ccggtgaaca acttatatat

[0033] ggaggttggc atggtcaaga ctatggaaat atagtcacct ttgatgctgt gcctgcaaat

[0034] gatcaggcca cggcatatga gctagtcctg acagttaatc aggaccaagc tccggcatca

[0035] cctgggaacc ggcgactctt tcaggtccgt ccaacgcaaa acgggcagca actcaacctg

[0036] aacaagctca attacaatgc tacatattcg ttcttgaggc ttggttcgga tggtaatctt

[0037] aaggcttata cttactatga ccaagtgagt tacttaaaat gggaagaaag ttttgctttc

[0038] ttttcaagtt acttcattag ggaatgtgca ttgccatcca aatgtggtgc atatggttta

[0039] tgtaacaagg gaatgtgcgt ggcgtgcccg agccctacag ggttgttggg ctggagcgaa

[0040] agatgtgccc cgcctaagcc gcctccatgt gggaaaggag ggaaggttga ttactacaaa

[0041] atagtgggtg tggagcactt tctaagtcca tatatggatg atggtaaagg tccaatgaag

[0042] gttggggaat gtagagataa atgtaacaag gattgcaagt gtttaggatt tttctacaag

[0043] gaagatacct ctaaatgctt gctagcacca ttgcttggta cactcatcaa acatgtcaat

[0044] actactgctg tgggatatat caagtatgca agatag.

[0045] exist Figure 1 In the figure, I-guiwei: pericarp of Guiwei litchi inoculated with Downy mildew; I-heiye: pericarp of Heiye litchi inoculated with Downy mildew. The relative expression level of LcCLP1 gene was verified by qRT-PCR using the pericarp of Heiye and Guiwei litchi inoculated with Downy mildew. The results were basically consistent with the results of transcriptome analysis.

[0046] The leaves of different litchi genotypes were used as materials and inoculated with anthracnose to analyze the changes in the expression level of the gene corresponding to anthracnose infection, such as Figure 2 It was found that in the material ZW107 with strong resistance to anthrax, the relative expression level of this gene was high and the expression level increased with the severity of the disease; in the material ZW72 with poor resistance to anthrax, the relative expression level of this gene also increased with the delay of release time, but the relative expression level was low and the change was small.

[0047] The gene was cloned and it was found that the DNA sequence and amino acid sequence of the gene were different in different genotypes. The nucleic acid sequence and amino acid sequence of the susceptible litchi resources 'Guiwei' and ZW72 were consistent, while the disease-resistant resources 'Heiye' and ZW107 were different from the former two. There were 15 differences in nucleic acid sequence between 'Heiye' and the former two, and 3 differences in nucleic acid sequence between ZW107 and the former two. In terms of amino acid sequence, there were 8 differences in amino acid sequence between 'Heiye' and the former two, and 3 differences in amino acid sequence between ZQ107 and the former two. See Figure 3 , Figure 4 .

[0048] It can be seen that this gene has a broad-spectrum disease resistance function and can be used in biotechnology breeding research, creating disease-resistant resources, and developing molecular marker-assisted breeding technology. This gene has a broad-spectrum resistance. If the creation of disease-resistant resources is used, the overexpression of a gene can achieve the effect of defending against two pathogens (litchi anthracnose and downy mildew); this gene has a broad-spectrum resistance. In molecular marker-assisted breeding, the workload can be reduced by 50% when selecting disease-resistant resources in the early stage.

[0049] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A litchi downy mildew resistance gene LcCLP1, characterized in that: The gene sequence of gene LcCLP1 is as follows: atggctgctt ttcatgtaat tttagtccat agcaccctcc tcagcctagc catcaccgga actctatccc aagtcccaac taaccaaacc ttcaaattca taaaccaagg tgaattcgga gacagaataa tcgaatatga cgccagttac cgtgtgatcc gaaacaatgt ttacacgttc tacacattcc catttcgcct gtgtttctac aacaccaccc ctaatgccta catttttgct attagggcag gcatccctaa tgatgaaagc ctcatgcgct gggtttggga cgccaaccgc aacaacccgg tacgtgagaa tgccaccctc acctttggcc ggaacggcaa cttggtcctg gcggccgccg atggtcgcgt ggcctggcaa acaaacacgg ccaacaaggg ggtcactggc atcaagttgt taccaaatgg taacctagtg ttgcatgaca aaaatgggaa atttatatgg gaaagttttg attaccctag tgatacttta ttggtgggcc agtcagtcaa aatcaacggt cgtaacaaac ttgttggtcg tacgtctgac atgattggct ctgatggacg gtacagtatt gtgctggatc ataaagggtt caccatgtat ttaaataatt ccggtgaaca acttatatat ggaggttggc atggtcaaga ctatggaaat atagtcacct ttgatgctgt gcctgcaaat gatcaggcca cggcatatga gctagtcctg acagttaatc aggaccaagc tccggcatca cctgggaacc ggcgactctt tcaggtccgt ccaacgcaaa acgggcagca actcaacctg aacaagctca attacaatgc tacatattcg ttcttgaggc ttggttcgga tggtaatctt aaggcttata cttactatga ccaagtgagt tacttaaaat gggaagaaag ttttgctttc ttttcaagtt acttcattag ggaatgtgca ttgccatcca aatgtggtgc atatggttta tgtaacaagg gaatgtgcgt ggcgtgcccg agccctacag ggttgttggg ctggagcgaa agatgtgccc cgcctaagcc gcctccatgt gggaaaggag ggaaggttga ttactacaaa atagtgggtg tggagcactt tctaagtcca tatatggatg atggtaaagg tccaatgaag gttggggaat gtagagataa atgtaacaag gattgcaagt gtttaggatt tttctacaag gaagatacct ctaaatgctt gctagcacca ttgcttggta cactcatcaa acatgtcaat actactgctg tgggatatat caagtatgca agatag.

2. The method for obtaining the litchi downy mildew resistance gene LcCLP1 according to claim 1, characterized in that: The steps include: S1: Select fruits of litchi varieties that are stable susceptible to Downy Phytophthora and litchi varieties that are resistant to Downy Phytophthora as samples; S2: Using transcriptome sequencing to analyze samples at different time points after infection with Downy Phytophthora, a group of differentially expressed genes were obtained; S3: obtaining a disease resistance-related gene CLP from the differentially expressed genes obtained in step S2; performing transcription, and based on the transcriptome data, cloning the full-length gene sequence and naming it LcCLP1; S4: The relative expression level of the LcCLP1 gene was verified by qRT-PCR using the peels of disease-resistant and susceptible litchi varieties after inoculation.

3. The method for obtaining the litchi downy mildew resistance gene LcCLP1 according to claim 2, characterized in that: The differentially expressed genes described in step S3 have a higher expression level in the pericarp of varieties resistant to downy mildew, and a lower expression level in the pericarp of varieties susceptible to the disease.

4. The method for obtaining the litchi downy mildew resistance gene LcCLP1 according to claim 2, characterized in that: In the step S1, the susceptible litchi variety is the 'Guiwei' litchi variety, and the disease-resistant litchi variety is the 'Heiye' litchi variety.

5. The use of the litchi downy mildew resistance gene LcCLP1 according to claim 1, characterized in that: The encoded protein is obtained through the litchi downy mildew resistance gene LcCLP1.

6. The use of the litchi downy mildew resistance gene LcCLP1 according to claim 1, characterized in that: The litchi downy mildew resistance gene LcCLP1 is used in litchi breeding and defense against litchi anthracnose and downy mildew.

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